Publications

• Characteristics of major Escherichia coli reductases involved in aerobic nitro and azo reduction.

  Mercier C., Chalansonnet V., Orenga S., Gilbert C.

  <h4>Aims</h4>Escherichia coli is able to reduce azo compounds such as methyl red (MR) and nitro compounds such as 7-nitrocoumarin-3-carboxylic acid (7NCCA). The aim of this study was to clarify the specificity of the major E. coli reductases.<h4>Methods and results</h4>Enzymatic assays with pure e ... >> More

  <h4>Aims</h4>Escherichia coli is able to reduce azo compounds such as methyl red (MR) and nitro compounds such as 7-nitrocoumarin-3-carboxylic acid (7NCCA). The aim of this study was to clarify the specificity of the major E. coli reductases.<h4>Methods and results</h4>Enzymatic assays with pure enzymes obtained after cloning, overproduction and purification under native or denaturing conditions were performed on three enzymes: AzoR, NfsA and NfsB. Their dependence on putative cofactors such as flavin mononucleotide (FMN), NADH and NADPH was studied as well as the reductase capacity of E. coli mutants depleted for one, two or three of the corresponding genes.<h4>Conclusions</h4>AzoR was able to reduce both MR and 7NCCA, whereas NfsA and NfsB could only reduce the nitro compound. AzoR and NfsB were strictly FMN dependent in contrast to NfsA. At a low oxygen concentration, the three proteins were not mandatory for azo reduction and nitro reduction, but in optimal aerobic conditions, azoR was essential for MR reduction, and an nfsA/nfsB combination was important for 7NCCA reduction. Overexpression of azoR gene was able to compensate for the loss of nfsA and nfsB under aerobic conditions.<h4>Significance and impact of study</h4>These data provide new insights into the substrate specificity of major E. coli nitroreductases and demonstrate that oxygen is an important parameter to take into account in studies of nitroreductase activity. << Less

  J. Appl. Microbiol. 115:1012-1022(2013) [PubMed] [EuropePMC]

• Expansion of substrate specificity and catalytic mechanism of azoreductase by X-ray crystallography and site-directed mutagenesis.

  Ito K., Nakanishi M., Lee W.C., Zhi Y., Sasaki H., Zenno S., Saigo K., Kitade Y., Tanokura M.

  AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several ... >> More

  AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme. AzoR in its reduced form revealed a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The crystal structure of oxidized AzoR in a different space group and the structure of the enzyme in complex with the inhibitor dicoumarol were also determined. These structures indicate that the formation of a hydrophobic part around the isoalloxazine ring is important for substrate binding and an electrostatic interaction between Arg-59 and the carboxyl group of the azo compound causes a substrate preference for methyl red over p-methyl red. The substitution of Arg-59 with Ala enhanced the Vmax value for p-methyl red 27-fold with a 3.8-fold increase of the Km value. This result indicates that Arg-59 decides the substrate specificity of AzoR. The Vmax value for the p-methyl red reduction of the R59A mutant is comparable with that for the methyl red reduction of the wild-type enzyme, whereas the activity toward methyl red was retained. These findings indicate the expansion of AzoR substrate specificity by a single amino acid substitution. Furthermore, we built an authentic model of the AzoR-methyl red complex based on the results of the study. << Less

  J. Biol. Chem. 283:13889-13896(2008) [PubMed] [EuropePMC]

• Crystallization and preliminary X-ray analysis of azoR (azoreductase) from Escherichia coli.

  Ito K., Nakanishi M., Lee W.-C., Sasaki H., Zenno S., Saigo K., Kitade Y., Tanokura M.

  AzoR (azoreductase), an FMN-dependent NADH-azo compound oxidoreductase from Escherichia coli, has been crystallized in the presence of FMN by the sitting-drop vapour-diffusion method using 2-propanol as a precipitant. AzoR catalyzes the reductive cleavage of azo groups. The crystals were found to ... >> More

  AzoR (azoreductase), an FMN-dependent NADH-azo compound oxidoreductase from Escherichia coli, has been crystallized in the presence of FMN by the sitting-drop vapour-diffusion method using 2-propanol as a precipitant. AzoR catalyzes the reductive cleavage of azo groups. The crystals were found to diffract X-rays to beyond 1.8 A resolution using a synchrotron-radiation source. The crystals belonged to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 92.2, c = 51.9 A. The crystals are expected to contain one subunit of the homodimer in the asymmetric unit (VM = 2.6 A3 Da(-1)) and to have a solvent content of 51.6%. Data sets were also collected from heavy-atom derivatives for use in phasing. As a result, crystals soaked in a solution containing K2PtCl4 for 23 d were found to be reasonably isomorphous to the native crystals and the presence of Pt atoms could be confirmed. The data sets from the native crystals and the K2PtCl4-derivatized crystals are being evaluated for use in structure determination by single isomorphous replacement with anomalous scattering. << Less

  Acta Crystallogr. F 61:399-402(2005) [PubMed] [EuropePMC]

• Putative ACP phosphodiesterase gene (acpD) encodes an azoreductase.

  Nakanishi M., Yatome C., Ishida N., Kitade Y.

  An FMN-dependent NADH-azoreductase of Escherichia coli was purified and analyzed for identification of the gene responsible for azo reduction by microorganisms. The N-terminal sequence of the azoreductase conformed to that of the acpD gene product, acyl carrier protein phosphodiesterase. Overexpre ... >> More

  An FMN-dependent NADH-azoreductase of Escherichia coli was purified and analyzed for identification of the gene responsible for azo reduction by microorganisms. The N-terminal sequence of the azoreductase conformed to that of the acpD gene product, acyl carrier protein phosphodiesterase. Overexpression of the acpD gene provided the E. coli with a large amount of the 23-kDa protein and more than 800 times higher azoreductase activity. The purified gene product exhibited activity corresponding to that of the native azoreductase. The reaction followed a ping-pong mechanism requiring 2 mol of NADH to reduce 1 mol of methyl red (4'-dimethylaminoazobenzene-2-carboxylic acid) into 2-aminobenzoic acid and N,N'-dimethyl-p-phenylenediamine. On the other hand, the gene product could not convert holo-acyl carrier protein into the apo form under either in vitro or in vivo conditions. These data indicate that the acpD gene product is not acyl carrier protein phosphodiesterase but an azoreductase. << Less

  J. Biol. Chem. 276:46394-46399(2001) [PubMed] [EuropePMC]